Project Summary Complex organisms protect themselves against threats via inflammation, which is in part controlled by brain pathways involving the neurotransmitter orexin. In sepsis, inflammation becomes dysregulated and organ dysfunction develops. We have shown that orexinergic activity is attenuated in experimental sepsis (cecal ligation and puncture (CLP) in mice). The main objectives of this project are to identify how sepsis-induced attenuation of orexinergic activity effects organ function, alters the host inflammatory response and contributes to mortality, and to determine if correcting decreased orexinergic activity reverses these changes. We will address these objectives via three Specific Aims. Specific Aim 1. Determine the effects of CLP-induced loss of orexinergic activity on organ function and pathology and on survival. Examine the contribution of the autonomic nervous system to these changes. Approach: We will reverse the CLP-induced loss of orexinergic activity with ICV orexin injection and examine how this alters cardiac, pulmonary, hepatic, renal and immune function, and survival. We will use peripheral ?-adrenergic and muscarinic blockade to identify the contribution of the autonomic nervous system. Further, we will determine if an ICV orexin infusion delivered at several different time points improves 14-day survival. Specific Aim 2. Determine the effects of the CLP-induced loss of orexinergic activity on brain cholinergic activity and on the inflammatory reflex. Approach: We will reverse the CLP-induced loss of orexinergic activity with an ICV injection of orexin and determine how this effects basal forebrain muscarinic and inflammatory reflex activity. To accomplish this we will examine the effects of an ICV orexin injection on 1) co-localization of choline-acetyltransferase (ChAT) and c-fos immunostaining, indicative of cholinergic activity, in appropriate brain sections, 2) serum levels of TNF?, IL-6 and HMGB1 and 3) TNF?, IL-6 and HMGB1 levels in the medium of harvested, stimulated splenocytes. . Specific Aim 3. Determine the effects of CLP-induced loss of brain cholinergic activity on orexinergic activity and, as a result, on alterations in HR, RR, T and pituitary hormone secretion. Approach: We will reverse the CLP-induced loss of brain cholinergic activity with xanomeline, a central M1 agonist, or ICV nicotine, and determine if orexinergic activity and HR, RR, T and pituitary hormone secretion are restored. We will then examine the effects of the orexin antagonist almorexant Demonstrating that CLP-induced changes in the orexinergic system modulate organ dysfunction, inflammatory responses and survival could re-direct the investigative and therapeutic paradigm to focus on the brain as a primary driver of sepsis, overcoming a critical barrier to progress. Demonstrating that we can alter these effects by enhancing brain cholinergic activity could provide a new, clinically viable therapeutic avenue for use in this deadly, highly prevalent disorder.